Part:BBa_K3019007

Pre-Ost1-pro-α-factor signal GLCI

Pre-Ost1-pro-α-factor signal fused to glcI N-terminal domain.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 558
  Illegal AgeI site found at 1639
  
• 1000
  COMPATIBLE WITH RFC[1000]

We have tested Ost1-Glc1 strain in time-lapse microscopy experiments following the cells for up to 20 hours after induction of glucanase genes from the estradiol-activated promoter. The experiments revealed that an increase in cell size could be detected at around 5 hours after the start of glucanase induction. The origin of the slow responsiveness could be that the estradiol induction in general shows relatively slow target protein accumulation dynamics [1]. In the presence of estradiol Ost1-Glc1 cells have abnormal morphology: the cells are enlarged, have swelled vacuoles, some are elongated, possibly due to cell wall defects (Fig. 1).


Figure 1. Time-lapse microscopy of six yeast strains grown in glucanase expression non-inductive (- panel, without ß-estradiol) or inductive (+ panel, with ß-estradiol) conditions. Brightfield (BF), GFP fluorescence and merged channel images are shown. Minutes indicate the time from ß-estradiol addition. Different strains contain different glucanase genes expressed from LexA promoter and are represented in following order: Pre-Ost1-pro-α-factor-Glc1 fusion. In the later stages of the experiment, at around 8 hours from the start of the induction, cells start dying rapidly. There were cells with defined lysis, in which case the release of intracellular material could be detected (see Movies 1).
Regrettably, we were unable to get all cells lysed even after 20 hours of induction. The most probable explanation is that the secretion signals we used were not very effective. 20 hours after the start of glucanase induction, about 7-13% of cells had died, whereas the percentage of dead cells was around 1% or less in the positions without estradiol (Fig. 2).


Figure 2. The effect of glucanase expression on cell viability. The percentage of dead cells was counted from time-lapse microscopy experiments 20 hours after the start of experiment. The experiment was performed with six strains expressing different glucanases from the estradiol-responsive promoter. The plot shows the percentage of dead cells in the presence or absence of estradiol in the medium.

REFERENCES

[1] Ottoz DSM, Rudolf F, Stelling J (2014) Inducible, tightly regulated and growth condition-independent transcription factor in Saccharomyces cerevisiae. Nucleic Acids Res 42:e130. https://doi.org/10.1093/nar/gku616

https://static.igem.org/mediawiki/parts/4/4d/T--Tartu_TUIT--ost_gl.mp4